By continuing to browse this site you agree to us using cookies as described in About Cookies
Notice: Wiley Online Library will be unavailable on Saturday 7th Oct from 03.00 EDT / 08:00 BST / 12:30 IST / 15.00 SGT to 08.00 EDT / 13.00 BST / 17:30 IST / 20.00 SGT and Sunday 8th Oct from 03.00 EDT / 08:00 BST / 12:30 IST / 15.00 SGT to 06.00 EDT / 11.00 BST / 15:30 IST / 18.00 SGT for essential maintenance. Apologies for the inconvenience.
Present address: Graduate School of Bioscience, Nagahama Institute of Bio-Science and Technology, Tamura-cho 1266, Nagahama, Shiga 526-0829, Japan. Correspondence regarding medaka to S. Yuba, email@example.com; regarding zebrafish to K. Hatta, firstname.lastname@example.org; regarding Arabidopsis to H. Urawa, email@example.com.
Heat shock promoters are powerful tools for the precise control of exogenous gene induction in living organisms. In addition to the temporal control of gene expression, the analysis of gene function can also require spatial restriction. Recently, we reported a new method for in vivo, single-cell gene induction using an infrared laser-evoked gene operator (IR-LEGO) system in living nematodes (Caenorhabditis elegans). It was demonstrated that infrared (IR) irradiation could induce gene expression in single cells without incurring cellular damage. Here, we report the application of IR-LEGO to the small fish, medaka (Japanese killifish; Oryzias latipes) and zebrafish (Danio rerio), and a higher plant (Arabidopsis thaliana). Using easily observable reporter genes, we successfully induced gene expression in various tissues in these living organisms. IR-LEGO has the potential to be a useful tool in extensive research fields for cell/tissue marking or targeted gene expression in local tissues of small fish and plants.
Heat shock response is one of the major stress responses and is widely conserved in all organisms, from bacteria to higher plants and vertebrates (Morimoto & Santoro 1998; Feder & Hofmann 1999; Basu et al. 2002). Heat stress initiates transcription of heat shock proteins (HSP) downstream of a promoter, known as the heat shock (hsp) promoter. As hsp promoters can be easily induced, they have often been used for gene induction at discretionary timing in transgenic organisms.
While whole-body heating is the conventional method to heat shock transgenic organisms, cell- or tissue-level induction is required for detailed, spatial gene analysis. Single-cell gene induction using heat shock promoters has been reported in Caenorhabditis elegans (Stringham & Candido 1993), Drosophila (Halfon et al. 1997) and in zebrafish (Halloran et al. 2000; Sato-Maeda et al. 2006). In those studies, a cell ablation dye laser system (wavelength of 440 nm) was converted into an effective tool to activate gene expression by reducing the laser power. Recently, a new method known as the infrared laser-evoked gene operator (IR-LEGO), was developed for in vivo, single-cell gene induction in C. elegans (Kamei et al. 2009). IR-LEGO uses an IR laser (wavelength of 1480 nm) to heat the target cells as this wavelength matches the combination of symmetric and asymmetric hydroxyl group (OH) stretching modes of water. By using an IR laser, the system avoids generating photochemical reactions that could damage the cells. As a result, the IR-LEGO system achieves a high efficiency and reproducibility of gene induction without cellular damage.
Arabidopsis thaliana is a well-researched plant genus (Bowman 1994; Meyerowitz & Somerville 1994) that has been used for heat shock-induced gene expression studies (Gallois et al. 2004; Heidstra et al. 2004). Although these reports revealed the importance of intercellular communication in the maintenance and differentiation of stem cells, the targeted gene was expressed in many cells and therefore the detailed mechanisms of communication could not be determined. Using a single cell gene induction system such as IR-LEGO might provide insight into the mechanisms of highly controlled intercellular communication and prompted us to evaluate the IR-LEGO system in A. thaliana.
The small fish, medaka (Oryzias latipes) and zebrafish (Danio rerio), are also useful model animals to study developmental biology as their genome sequences are readily available, and a variety of molecular biology and transgenic techniques have been established in these organisms (Wittbrodt et al. 2002; Furutani-Seiki & Wittbrodt 2004). Here, we report the first application of IR-LEGO in a higher plant, A. thaliana, and the vertebrates, medaka and zebrafish, and demonstrate the versatility of this system for controlled gene induction.
Materials and methods
Animals and plant
The transgenes used to construct the transgenic organisms are shown in Figure 1(A). In medaka, the medaka hsp70.1 promoter and a green fluorescent protein (GFP) variant, Venus, was used as a reporter gene. The line of hsp70.1::Venus carries a transgene, which codes for the Venus protein fused with luciferase; however, luciferase activity was not assayed in the present study.
In zebrafish, the Gal4/UAS system was used. The Gal4 gene was positioned downstream of the hsp70 promoter cloned from the zebrafish genome. In addition, a fluorescent protein, Kaede, which can alter its fluorescent color by photoconversion when irradiated with UV-light, was used as a reporter (Ando et al. 2002). The Kaede gene was positioned downstream of the UAS promoter. Double transgenic fish, hsp70::Gal4 and UAS::Kaede, were obtained by the crossing of these two lines, as reported previously (Hatta et al. 2006). Fish embryos and larvae were cultured in normal culture medium at 27°C before and after laser irradiation. The zebrafish embryos were morphologically staged according to the scheme described previously (Kimmel et al. 1995).
In A. thaliana, the hsp18.2 promoter from A. thaliana (Takahashi & Komeda 1989) and the β-glucuronidase (GUS) gene were used as a reporter (Jefferson et al. 1987). Seeding condition is described in Supporting Information. Establishment of the transgenic line for hsp18.2::GUS was described previously (Takahashi et al. 1992).
IR-LEGO microscope system
The optical system of the IR-LEGO microscope (Fig. 1B) was slightly modified from that described in our previous report (Kamei et al. 2009). An IR-LEGO optical unit (special order item; Sigma-Koki, Japan) was used for this system. Two mono-coated objectives (UApo340 20×/0.75 UV and UApo340 40×/0.90 UV, Olympus, Japan; Supporting Information) were used for irradiation of the targets with the IR laser.
IR laser irradiation and observation of induced gene expression
During the trials of IR laser irradiation of the targets, fish and seedlings were embedded in a 3% methylcellulose solution for stable positioning of the targets. If necessary, the medaka larvae and zebrafish embryos were anesthetized with 0.02% MS222 (M0387; Sigma) before embedding (Aramaki & Hatta 2006). To prevent pigmentation by melanin synthesis, the zebrafish embryos were grown in 0.003% 1-phenyl-2-thiourea. Irradiation was carried out at room temperature (22°C). After IR irradiation, target organisms were kept at normal culture conditions for 6–40 h before the samples were observed. Venus and Kaede expression in the fish was observed by fluorescence microscopy with a fluorescent filter set for GFP (Supporting Information). To visualize target gene expression in A. thaliana we used the GUS staining (Supporting Information). After staining, the seedlings were embedded in chloral hydrate/glycerol/water (8:1:2).
IR-LEGO operation in medaka
A transgenic medaka (hsp70.1::Venus) line that expresses luciferase and Venus under the control of the medaka hsp70.1 promoter (Fig. 1A) was used for in vivo gene induction by the IR-LEGO system. In this transgenic line, the medaka hsp70.1 promoter has been shown to be almost entirely inactive in all tissues at the embryonic and early larval stage under normal culture conditions, with the exception of the lens, which does express Venus (S. Oda, unpubl. data, 2009).
Using this transgenic larva (14 days postfertilization), three developing oral teeth in the left half of the lower jaw of a living larva were irradiated using the IR laser (20–25 mW for 0.5–1.0 s). The three teeth became brightly fluorescent 18 h after the irradiation, while the non-irradiated teeth in the right half of the lower jaw showed no fluorescence (Fig. 2A–D). The expression of Venus in the teeth appeared to be power-dependent and it was found that irradiation with a laser of 20 mW for 0.5 s (tooth 1 in Fig. 2A) was ideal for inducing gene expression in a single tooth. Venus expression could be clearly observed in the dental papilla and pulp of the developing teeth (Fig. 2D).
To confirm the specificity and reproducibility of IR-LEGO for inducing Venus expression in other targeted organs, a transgenic medaka larva was irradiated at four points in the left half of its pineal gland (Fig. 2E–H). Again, Venus was only specifically induced in the left half of the pineal gland, 18 h after irradiation. Another larva was irradiated with IR laser at two points in its dorsal skeletal muscle (Fig. 2I–K). After 18 h, Venus fluorescence was clearly observed in the targeted regions with no apparent cellular damage.
The final organ in which, we tried to induce heat shock-mediated gene expression by IR-LEGO was the heart. As the heart was continually beating, it was somewhat difficult to irradiate the ventricular wall; however, it was successfully irradiated with three pulses of the IR laser using a power of 20 mW for 0.5 s. During the irradiation, the heart often stopped beating or showed fibrillation, but resumed normal beating within a few seconds after the irradiation. After 18 h, Venus was successfully induced in the ventricular wall of the heart (Fig. 2L–N) which beat normally (Supporting Information movie).
IR-LEGO operation in zebrafish
For the zebrafish, a Gal4/UAS system was used for reporter gene expression (Fig. 1A). The zebrafish hsp70 promoter was almost completely inactive at the embryonic stage under normal culture conditions, but the lens and a certain group of anterior spinal neurons often expressed Kaede even without heat shock (Hatta et al. 2006).
The skeletal muscle and retinal cells were irradiated with the IR laser using 20× or 40× objectives and a power range of 10–30 mW for 1 s. The typical expression patterns of the marker, Kaede, are shown in Figure 3. The somite cells of embryos 28 h postfertilization (28 hpf), irradiated with the IR laser (20 mW for 1 s, 40× objective), only expressed Kaede in the targeted somite cell, approximately 4 h after irradiation (Fig. 3A–C). In another embryo at 18 hpf, we attempted to irradiate a cell in the pronephric duct. However, after 25 h, the green fluorescence of Kaede was expressed in three distinct tissues: the skin, muscle, and pronephric duct (Fig. 3D–F). We believe the cells were located at the same position in the x-y plane when they were irradiated, and thereafter their relative positioning was separated during development of the embryo.
Using an embryo at 24 hpf, we also irradiated four points on the notochord using an IR laser of various powers and durations. After 20 h, Kaede expression was observed specifically in the targeted regions (Fig. 3G–I). Finally, two retinal cell groups facing each other across the lens were irradiated. Kaede expression in the retina was confirmed in both groups of cells at 40 h post-irradiation. Subsequently, we subjected one group of retinal cells to Kaede photoconversion using ultra-violet light to demonstrate availability of further subset labeling. The targeted retinal cells changed from green to red, enabling us to clearly distinguish individual cells and their projection through the optic nerve (Fig. 3J–L).
IR-LEGO operation in Arabidopsis
To demonstrate the versatility of the IR-LEGO, we also examined its applicability in a transgenic A. thaliana line (hsp18.2-GUS) (Fig. 1A). The A. thaliana hsp18.2 promoter was constitutively active in the bundle sheath and hypocotyl of 8-day-old seedlings under normal cultivation conditions, but was inactive in the lateral root tips (Fig. 4A–B). By whole-plant heating, GUS expression was induced in all tissues including the lateral root tip (Fig. 4A–D). Thus, we irradiated the laser to the epidermal cells, which were located at the inside of the root cap cell layer of the lateral root tip.
Four cells located in a close proximity were irradiated with the IR laser at 11 mW for 1 s. After 6 h, the seedling was stained using the GUS assay and strong GUS expression was detected in the four targeted cells; however, a moderate GUS signal was also detected in the immediate vicinity of the target cells (Fig. 4E–F). In an attempt to reduce the diffusion of the reaction product, after irradiation of a single root cell we carried out a different staining technique (Sessions et al. 1999). Using this method, only the targeted cell exhibited GUS staining (Fig. 4G), albeit somewhat weaker than the staining observed in Figure 4E.
In the present study, we have demonstrated the applicability of the IR-LEGO system for use in fish (medaka and zebrafish), and a higher plant (A. thaliana), by using native heat shock promoters. For the zebrafish and A. thaliana, we were able to use the heat shock promoters hsp70 and hsp18.2, respectively, which have been previously shown to be effectively activated by heat shock (Takahashi & Komeda 1989; Takahashi et al. 1992; Halloran et al. 2000; Sato-Maeda et al. 2006). In medaka, however, the two prior studies using heat shock-inducible transgenic medaka were carried out with either the zebrafish hsp70 promoter (Grabher & Wittbrodt 2004) or an artificial promoter (Bajoghli et al. 2004). Here we constructed a transgenic medaka line using the native medaka hsp70.1 promoter upstream of the reporter gene (S. Oda, unpubl. data, 2009). All promoters in the present study work effectively in many tissues using the IR-LEGO system.
We originally fixed the irradiation conditions based on the data from the initial report of IR-LEGO in nematodes (11 mW for 1 s) (Kamei et al. 2009), although we used different magnification objectives with different degrees of IR transmittance (Supporting Information). During the process of determining the optimal irradiation conditions for single cell gene induction, we found that IR irradiation could induce multi-cellular gene expression. Single cell gene induction is required for analyses related to intercellular communication, while multi-cellular gene induction may also be useful for gene analysis at the organ/tissue level. We think this local (multi-cellular) gene induction would be difficult to accomplish using the visible laser system.
For medaka and zebrafish, we found that in general the optimal laser conditions for the various tissues examined was between 15 and 25 mW for a duration of 0.5–1.0 s, while it was between 11 and 15 mW for 1–2 s in A. thaliana. However, within this range there exists an ideal laser power and duration to induce gene expression, which must be determined empirically for each specific tissue or cell type. This difference may be ascribable to the depth of the target cell in the embryo since the IR has a high absorbance factor in water, the main constituent of cells. We should also take into consideration the depth of a target cell when we carry out IR irradiation. A rough estimation of IR absorbance for a depth of 100 μm is 0.25. In other words, power reaching a target 100 μm deep is 75% of the initial power at the focus. We confirmed that cells located a depth of approximately 150-μm from the surface expressed gene products by IR-laser irradiation (data not shown).
The optimal laser power needed to induce gene expression in A. thaliana was somewhat lower than the power need for medaka or zebrafish. A possible explanation might relate to differences in the cell wall structure between plants and animals, because thick plant cell walls may reduce the diffusion of heat and thereby lower the laser power needed to induce a heat shock response.
One of the main applications of IR-LEGO will be for gene function analysis in vivo, when the spatial and temporal control of gene expression is required. Using IR-LEGO to induce single-cell gene expression can also solve problems originating from gene expression in non-target cells as often occurs with other techniques, such as mosaic analysis. In the present study, we demonstrated the specificity and versatility of the IR-LEGO system using the model organisms, medaka, zebrafish, and A. thaliana. IR-LEGO can be applied to all transparent organisms in which transgenic technology is available. Our results clearly indicate the potential of the IR-LEGO system for functional gene analysis by exchanging the reporter with a target gene in the transgenic construction. When combined with other techniques, such as site-specific recombination, IR-LEGO could be a very powerful tool for gene analysis and developmental studies of various species.
We thank S. Takagi and M. Suzuki (Nagoya Univ.) for introducing the technique of IR-LEGO; S. Kanamura, S. Aramaki, and laboratory members of the University of Hyogo for help in caring for the zebrafish lines; S. Hayashi for encouraging KH; Y. Morita and Y. Jozaki for help in caring for the medaka; H. Ohmiya (Sigma-Koki, Saitama, Japan) for technical advice regarding the optics used; and M. Yabuuchi (Olympus, Tokyo, Japan) for technical advice regarding objectives. We also thank the National Bio Resource Project Medaka for providing the hatching enzyme. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (KH: 15300117 and SO: 17659062); by the “Ground-based Research Program for Space Utilization” promoted by the Japan Space Forum (SO, HM, TTo, and YK); by a Grant-in-Aid for Creative Scientific Research from the Japan Society for the Promotion of Science (KO: No. 19GS0315), by a special research grant from the University of Hyogo (KH); and by the Hyogo Science and Technology Association (KH).
TD, MI, and HU contributed equally to this work. TD, TM and YK carried out the medaka experiments, MI and KH carried out the zebrafish experiments and HU carried out the Arabidopsis experiments. MI and YK evaluated the optical properties of the IR-LEGO devices. SN conceived and supported the medaka tooth experiment. TK, SO and HM established the medaka transgenic lines. TTa established the Arabidopsis transgenic line. TTo, KO, JS and SY coordinated the research. HU, KH and YK formulated the research and wrote the manuscript.